Indole derivatives as MCP-1 receptor antagonists

ABSTRACT

A compound of Formula I, 
                 
 
wherein: R 1  is hydrogen, halo, methyl, ethyl or methoxy; R 2  is hydrogen, halo, methyl, ethyl or methoxy; R 3  is a halo group, lower alkyl, lower alkenyl, lower alkynyl, alkoxy, trifluoromethyl, nitro, cyano, trifluoromethoxy, C(O)R 7 , or S(O) n R 7  where n is 0, 1 or 2 and R 7  is an alkyl group; R 4  is a halo, trifluoromethyl, methylthio, methoxy, trifluoromethoxy or lower alkyl, lower alkenyl or lower alkynyl or COR 8  where R 8  is lower alkyl; R 6  is hydrogen, halo, lower alkyl, lower alkenyl, lower alkynyl or COR 9  where R 9  is lower alkyl; provided that when R 1  is hydrogen, halo or methoxy, R 2  is hydrogen, halo, methyl, ethyl or methoxy, R 5  and R 6  are both hydrogen, and one of R 3  or R 4  is not halo or trifluoromethyl; or a pharmaceutically acceptable salt or prodrug thereof. These compounds have useful activity for the treatment of inflammatory disease, specifically in antagonizing an MCP-1 mediated effect in a warm-blooded animal such as a human being.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 of PCTapplication PCT/GB01/00074, filed Jan. 9, 2001, which claims priorityfrom Great Britain Application No. 0000625.4, filed Jan. 13, 2000, thespecifications of each of which are incorporated by reference herein.

The present invention relates to anti-inflammatory compounds that actvia antagonism of the CCR2 receptor, (also known as the MCP-1 receptor),leading inter alia to inhibition of Monocyte Chemoattractant Protein-1(MCP-1). These compounds contain an indole moiety. The invention furtherrelates to pharmaceutical compositions containing them, processes fortheir preparation, intermediates useful in their preparation and totheir use as therapeutic agents.

MCP-1 is a member of the chemokine family of pro-inflammatory proteinswhich mediate leukocyte chemotaxis and activation. MCP-1 is a C—Cchemokine which is one of the most potent and selective T-cell andmonocyte chemoattractant and activating agents known. MCP-1 has beenimplicated in the pathophysiology of a large number of inflammatorydiseases including rheumatoid arthritis, glomerular nephritides, lungfibrosis, restenosis (International Patent Application WO 94/09128),alveolitis (Jones et al., 1992, J. Immunol., 149, 2147) and asthma.Other disease areas where MCP-1 is thought to play a part in theirpathology are atherosclerosis (e.g. Koch et al., 1992, J. Clin. Invest.,90, 772-779), psoriasis (Deleuran et al., 1996, J. DermatologicalScience, 13, 228-236), delayed-type hypersensitivity reactions of theskin, inflammatory bowel disease (Grimm et al., 1996, J. LeukocyteBiol., 59, 804-812), multiple sclerosis and brain trauma (Berman et al.1996, J. Immunol., 156, 3017-3023). An MCP-1 inhibitor may also beuseful to treat stroke, reperfusion injury, ischemia, myocardialinfarction and transplant rejection.

MCP-1 acts through the CCR2 receptor. MCP-2 and MCP-3 may also act, atleast in part, through this receptor. Therefore in this specification,when reference is made to “inhibition or antagonism of MCP-1” or “MCP-1mediated effects” this includes inhibition or antagonism of MCP-2 and/orMCP-3 mediated effects when MCP-2 and/or MCP-3 are acting through theCCR2 receptor.

The applicants have found a class of compounds containing an indolemoiety which have useful inhibitory activity against MCP-1.International Patent Application, Publication No. WO 99/07351 disclosesa class of indoles with MCP-1 inhibitory activity. This application isbased on the surprising discovery that particular substituted 5-hydroxyindoles are MCP-1 inhibitors which possess unexpected and beneficialproperties with respect to potency and/or blood levels and/orbioavailability and/or solubility.

Accordingly, the present invention provides a compound of the formula(I):

wherein:

-   -   R¹ is hydrogen, halo, methyl, ethyl or methoxy;    -   R² is hydrogen halo, methyl ethyl or methoxy;    -   R³ is a halo group, lower alkyl, lower alkenyl lower alkynyl,        alkoxy, trifluoromethyl, nitro, cyano, trifluoromethoxy, C(O)R⁷,        or S(O)_(n)R⁷ where n is 0, 1 or 2 and R⁷ is an alkyl group;    -   R⁴ is a halo, trifluoromethyl, methylthio, methoxy,        trifluoromethoxy or lower alkyl, lower alkenyl or lower alkynyl;    -   R⁵ is hydrogen, halo, cyano, lower alkyl, lower alkenyl or lower        alkynyl or COR⁸ where R⁸ is lower alkyl;    -   R⁶ is hydrogen, halo, lower alkyl, lower alkenyl lower alkynyl        or COR⁹ where R⁹ is lower alkyl;    -   provided that when R¹ is hydrogen, halo or methoxy, R² is        hydrogen, halo, methyl, ethyl or methoxy, R⁵ and R⁶ are both        hydrogen, and one of R³ or R⁴ is chloro, fluoro, or        trifluoromethyl, then the other of R³ or R⁴ is not halo or        trifluoromethyl;    -   or a pharmaceutically acceptable salt or prodrug thereof.

In this specification the term “alkyl” includes both straight andbranched chain alkyl groups but references to individual alkyl groupssuch as “propyl” are for the straight chain version only. Similarly theterm “alkenyl” and “alkynyl” refers to straight or branched chainunsaturated moieties. Unless otherwise stated, alkyl groups suitablycontain from 1 to 10 carbon atoms, preferably from 1 to 6 carbon atomsand most preferably from 1 to 4 carbon atoms. Alkenyl and alkynyl groupssuitably contain from 2 to 10 carbon atoms, preferably from 2 to 6carbon atoms and most preferably from 2 to 4 carbon atoms. The prefix“lower” indicates that the group has up to 6 and preferably up to 4carbon atoms. The term “halo” refers to fluoro, chloro, bromo and iodo.

Preferably R¹ is hydrogen or halo such as chloro or fluoro, and mostpreferably hydrogen.

Preferably R² is hydrogen or halo such as chloro or fluoro and mostpreferably hydrogen.

Suitable examples of R³ include halo such as chloro, nitro or alkoxysuch as methoxy.

In one embodiment, R³ is trifluoromethyl and R⁴ is methylthio, methoxy,trifluoromethoxy, alkyl in particular methyl or alkynyl in particularethynyl.

Particular examples of R⁴ are halo such as chloro, alkyl such as methyl,alkoxy such as methoxy or trifluoromethoxy.

In another preferred embodiment R⁴ is halo such as chloro ortrifluoromethyl and R³ is alkyl, alkenyl, alkynyl, alkoxy, nitro, cyano,trifluoromethoxy, C(O)R⁷, or S(O)_(n)R⁷ where R⁷ and n are as definedabove, and R⁷ in particular is methyl or ethyl.

Preferably R⁵ is hydrogen.

Preferably R⁶ is hydrogen.

In a preferred aspect of the invention there is provided a compound offormula (IA):

-   -   or a pharmaceutically acceptable salt or prodrug thereof,        wherein R¹, R² are as defined above;    -   R³′ is alkyl, alkenyl, alkynyl, alkoxy, trifluoromethyl, chloro,        nitro, cyano, trifluoromethoxy, C(O)R⁷, or S(O)_(n)R⁷ where R⁷        is an alkyl group;    -   R⁴′ is halo, methylthio, methoxy, trifluoromethoxy or methyl        group;        provided that when R³′ is trifluoromethyl, R⁴′ is not        trifluoromethyl or halo.

Preferably R¹ and R² are hydrogen.

Preferably R³ is selected from methoxy, chloro or nitro.

Preferably R⁴ is selected from chloro, methyl methoxy ortrifluoromethoxy.

Preferred compounds of the invention include any one of the Exampleswhich are illustrated in Table 1.

TABLE 1

Compound No. R³ R⁴ 1 OCH₃ Cl 2 Cl OCH₃ 3 Cl CH₃ 4 NO₂ CH₃ 5 Cl OCF₃ 6NO₂ Cl 7 F CH₃ 8 —C≡CH Cl

The invention further relates to all tautomeric forms of the compoundsof formula (I).

It is also to be understood that certain compounds of the formula (I)can exist in solvated as well as unsolvated forms such as, for example,hydrated forms. It is to be understood that the invention encompassesall such solvated forms.

Compounds of formula (I) are inhibitors of monocyte chemoattractantprotein-1. In addition, they appear to inhibit RANTES induced chemotaxisRANTES (Regulated upon Activation Normal T-cell Expressed and Secreted)is another chemokine from the same family as MCP-1, with a similarbiological profile, but acting though the CCR1 receptor. As a result,theses compounds can be used to treat disease mediated by these agents,in particular inflammatory disease.

Suitable pharmaceutically acceptable salts of compounds of formula (I)include base salts such as an alkali metal salt for example sodium, analkaline earth metal salt for example calcium or magnesium, an organicamine salt for example triethylamine, morpholine, N-methylpiperidine,N-ethylpiperidine, procaine, dibenzylamine, N, N-dibenzylethylamine oramino acids for example lysine. In another aspect, where the compound issufficiently basic, suitable salts include acid addition salts such asmethanesulphonate, fumarate, hydrochloride, hydrobromide, citrate,maleate and salts formed with phosphoric and sulphuric acid. There maybe more than one cation or anion depending on the number of chargedfunctions and the valency of the cations or anions. A preferredpharmaceutically acceptable salt is a sodium salt.

Various forms of prodrugs are known in the art. For examples of suchprodrug derivatives, see:

-   a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and    Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et    al. (Academic Press, 1985);-   b) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and    Application of Prodrugs”, by H. Bundgaard p. 113-191 (1991);-   c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);-   d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285    (1988); and-   e) N. Kakeya, et al., Chem Pharm Bull, 32, 692 (1984).

Examples of such prodrugs are in vivo cleavable esters of a compound ofthe invention. An in vivo cleavable ester of a compound of the inventioncontaining a carboxy group is, for example, apharmaceutically-acceptable ester which is cleaved in the human oranimal body to produce the parent acid. Suitablepharmaceutically-acceptable esters for carboxy include C₁₋₆alkyl esters,for example methyl or ethyl; C₁₋₆alkoxymethyl esters, for examplemethoxymethyl; C₁₋₆alkanoyloxymethyl esters, for examplepivaloyloxymethyl; phthalidyl esters; C₃₋₈cycloalkoycarbonyloxyC₁₋₆alkylesters, for example 1-cyclohexylcarbonyloxyethyl;1,3-dioxolan-2-ylmethyl esters, for example5-methyl-1,3-dioxolan-2-ylmethyl; C₁₋₆alkoxycarbonyloxyethyl esters, forexample 1-methoxycarbonyloxyethyl; aminocarbonylmethyl esters and mono-or di-N-(C₁₋₆alkyl) versions thereof, for example N,N-dimethylaminocabonylmethyl esters and N-ethylaminocarbonylmethylesters; and may be formed at any carboxy group in the compounds of thisinvention. An in vivo cleavable ester of a compound of the inventioncontaining a hydroxy group is, for example, apharmaceutically-acceptable ester which is cleaved in the human oranimal body to produce the parent hydroxy group. Suitablepharmaceutically acceptable esters for hydroxy include C₁₋₆alkanoylesters, for example acetyl esters; and benzoyl esters wherein the phenylgroup may be substituted with aminomethyl or N-substituted mono- ordi-C₁₋₆alkyl aminomethyl, for example 4-aminomethylbenzoyl esters and4-N,N-dimethylamiomethylbenzoyl esters.

Another aspect of the present invention provides a process for preparinga compound of formula (I) or a pharmaceutically acceptable salt orprodrug thereof which process comprises:

-   a) reacting compounds of formula (II):    where R¹, R², R⁵ and R⁶ are as defined in relation to formula (I),    R^(a) is carboxy or a protected form thereof and R^(b) is hydrogen    or a suitable hydroxy protecting group, with a compound of formula    (III):    where R³ and R⁴ are as defined in relation to formula (I) and L is a    displaceable group; and thereafter if necessary-   i) converting a compound of the formula (I) into another compound of    the formula (I);-   ii) removing any protecting groups; or-   iii) forming a pharmaceutically acceptable salt or prodrug thereof.

Suitable values for L are for example, a halogeno or sulphonyloxy group,for example a chloro, bromo, methanesulphonyloxy ortoluene-4-sulphonyloxy group.

Compounds of formula (II) and (III) are suitably reacted together in aninert organic solvent such as N,N-dimethylformamide, dichloromethane oracetonitile in the presence of a base such as sodium hydroxide, sodiumhydride or potassium carbonate. Suitably the reaction is effected in thepresence of a phase transfer catalyst such as tetra-n-butylammoniumhydrogensulphate. Reaction times may range for 1-6 hours preferably for1-3 hours.

Moderate temperatures for example of 15-30° C., preferably 20-25° C. areemployed.

Compounds of formula (II) may be commercially available, or they may bemade by modification using known processes of commercially availablecompounds of formula (II). In particular, they may be prepared byreacting a compound of formula (IV):

where R¹, R⁵, R⁶ and R^(b) is as defined above with a compound offormula (V)

where R^(c) and R^(c′) are independently selected from C₁₋₄alkyl.

Compounds of formula (IV) and (V) are suitably reacted together underReissert reaction conditions such as in an inert solvent (such astetrahydrofuran), in the presence of a base (such as potassiumethoxide), at a temperature range of 15-30° C. preferably 20-25° C., for10-20 hours preferably 15-17 hours. The resulting compound is isolatedand dissolved in an alcohol such as ethanol and an organic acid (such asacetic acid) and a transition metal catalyst (such as 10% Pd/C) andcyclohexene is added. The mixture may then be heated at a temperature of60-120° C. preferably at 70-90° C. for 15-25 hours preferably 16-20hours to give a compound of formula (II) wherein R^(a) is —CO₂C₁₋₄alkyl.

Alternatively, compounds of formula (II) can be prepared by reacting acompound of formula (VI):

where R¹, R⁵, R⁶ and R^(b) are as defined above, with a compound offormula (VII):

where R^(d) is C₁₋₄alkyl.

Compounds of formula (VI) and (VII) are suitably reacted together underFischer conditions such as with an organic acid (such as acetic acid),in an alcohol (such as ethanol), at a temperature of 60-90° C.,preferably 75-85° C., for 1-5 hours, preferably 1-3 hours. The resultingcompound is mixed with a strong acid (such as polyphosphoric acid) andheated at 90-150° C. preferably 100-120° C., for 0.5-4 hours, preferably0.5-2 hours to give a compound of formula (II) in which R² is hydrogen.Then, if desired, R² can be optionally converted into another value ofR² as defined in formula (I) using techniques known in the literature.

In a preferred alternative, compounds of formula (II) are obtained bycyclisation of a compound of formula (VIII)

where R¹, R^(a), R^(b) and R² are as defined above.

Cyclisation may be effected by refluxing the compound in an organicsolvent such as xylene. Compounds of formula (VIII) are suitablyprepared by reacting a compound of formula (IX)

where R¹, R² and R^(b) are as defined above, with a compound of formula(X)

where R^(a) is as defined above. The reaction is suitably effected in anorganic solvent such as an alcohol, in particular methanol in thepresence of a base such as an alkali metal alkoxide, in particularsodium methoxide. Moderate temperatures of from −30 to 20° C. aresuitably employed.

Compounds of formula (III), (IV), (V), (VI), (VII), (VIII), (IX) and (X)are known or commercially available or are prepared by processes knownin the art by standard manipulation of commercially available or knownmaterials.

R^(c) and R^(d) are C₁₋₄alkyl. Preferably R^(c) and R^(d) are methyl orethyl.

It will also be appreciated that in some of the reactions mentionedherein it may be necessary/desirable to protect any sensitive groups inthe compounds. The instances where protection is necessary or desirableand suitable methods for protection are known to those skilled in theart. Thus, if reactants include groups such as carboxy or hydroxy it maybe desirable to protect the group in some of the reactions mentionedherein.

A suitable protecting group or a hydroxy group is, for example, an acylgroup, for example an alkanoyl group such as acetyl an aroyl group, forexample benzoyl, or an arylmethyl group, for example benzyl. Thedeprotection conditions for the above protecting groups will necessarilyvary with the choice of protecting group. Thus, for example, an acylgroup such as an alkanoyl or an aroyl group may be removed, for example,by hydrolysis with a suitable base such as an alkali metal hydroxide,for example lithium or sodium hydroxide. Alternatively an arylmethylgroup such as a benzyl group may be removed, for example, byhydrogenation over a catalyst such as palladium-on-carbon.

A suitable protecting group for a carboxy group is, for example, anesterifying group, for example a methyl or an ethyl group which may beremoved, for example, by hydrolysis with a base such as sodiumhydroxide, or for example a t-butyl group which may be removed, forexample, by treatment with an acid, for example an organic acid such astrifluoroacetic acid, or for example a benzyl group which may beremoved, for example, by hydrogenation over a catalyst such aspalladium-on-carbon.

The protecting groups may be removed at any convenient stage in thesynthesis using conventional techniques well known in the chemical art.

Some of the intermediates described herein may be novel, for exampleintermediates of the formula (II), and as such they are provided as afurther feature of the invention.

When a pharmaceutically-acceptable sale of a compound of formula (I) isrequired, it may be obtained, for example, by reaction of said compoundwith the appropriate acid (which affords a physiologically acceptableanion), or with the appropriate base (which affords a physiologicallyacceptable cation), or by any other conventional salt formationprocedure.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of the formula (I)as defined hereinbefore or a pharmaceutically acceptable salt or prodrugthereof, in association with a pharmaceutically acceptable excipient orcarrier.

The compositions of the invention may be in a form suitable for oral use(for example as tablets, lozenges, bard or soft capsules, aqueous oroily suspensions, emulsions, dispersible powders or granules, syrups orelixirs), for topical use (for example as creams, ointments, gels, oraqueous or oily solutions or suspensions), for administration byinhalation (for example as a finely divided powder or a liquid aerosol),for inhalation by insufflation (for example as a finely divided powder)or for paternal administration (for example as a sterile aqueous or oilysolution for intravenous, subcutaneous, intramuscular or intramusculardosing or as a suppository for rectal dosing).

The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

Suitable pharmaceutically acceptable excipients for a tablet formulationinclude, for example, inert diluents such as lactose, sodium carbonate,calcium phosphate or calcium carbonate, granulating and disintegratingagents such as corn starch or algenic acid; binding agents such asstarch; lubricating agents such as magnesium stearate, stearic acid ortalc; preservative agents such as ethyl or propyl p-hydroxybenzoate, andanti-oxidants, such as ascorbic acid. Tablet formulations may beuncoated or coated either to modify their disintegration and thesubsequent absorption of the active ingredient within thegastrointestinal track, or to improve their stability and/or appearance,in either case, using conventional coating agents and procedures wellknown in the art.

Compositions for oral use may be in the form of hard gelatin capsules inwhich the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules in which the active ingredient is mixed with water oran oil such as peanut oil, liquid parafin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finelypowdered form together with one or more suspending agents, such assodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin or condensation products of an alkylene oxide with fatty acids(for example polyoxyethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives (such as ethyl orpropyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid),colouring agents, flavouring agents, and/or sweetening agents (such assucrose, saccharine or aspartame).

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil (such as arachis oil, olive oil, sesame oil orcoconut oil) or in a mineral oil (such as liquid paraffin). The oilysuspensions may also contain a thickening agent such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set outabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Suitable dispersing or wetting agents andsuspending agents a exemplified by those already mentioned above.Additional excipients such as sweetening, flavouring and colouringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, or a mineral oil, such as for exampleliquid paraffin or a mixture of any of these. Suitable emulsifyingagents may be, for example, naturally-occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soya bean,lecithin, an esters or partial esters derived from fatty acids andhexitol anhydrides (for example sorbitan monooleate) and condensationproducts of the said partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such asglycerol, propylene glycol, sorbitol, aspartame or sucrose, and may alsocontain a demulcent, preservative, flavouring and/or colouring agent.

The pharmaceutical compositions may also be in the form of a sterileinjectable aqueous or oily suspension, which may be formulated accordingto known procedures using one or more of the appropriate dispersing orwetting agents and suspending agents, which have been mentioned above. Asterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parentally-acceptable diluent or solvent,for example a solution in 1,3-butanediol.

Suppository formulations may be prepared by mixing the active ingredientwith a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Suitable excipients include, forexample, cocoa butter and polyethylene glycols.

Topical formulations, such as creams, ointments, gels and aqueous oroily solutions or suspensions, may generally be obtained by formulatingan active ingredient with a conventional, topically acceptable, vehicleor diluent using conventional procedure well known in the art.

Compositions for administration by insulation may be in the form of afinely divided powder containing particles of average diameter of, forexample, 30μ or much less, the powder itself comprising either activeingredient alone or diluted with one or more physiologically acceptablecarriers such as lactose. The powder for insulation is then convenientlyretained in a capsule containing, for example, 1 to 50 mg of activeingredient for use with a turbo-inhaler device, such as is used forinsufflation of the known agent sodium cromoglycate.

Compositions for administration by inhalation may be in the form of aconventional pressurised aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may used and the aerosol deviceis conveniently arranged to dispense a metered quantity of activeingredient.

For further information on Formulation the reader is referred to Chapter25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will generally contain, for example, from 0.5mg to 2 g of active agent compounded with an appropriate and convenientamount of excipients which may vary from about 5 to about 98 percent byweight of the total composition. Dosage unit forms will generallycontain about 1 mg to about 500 mg of an active ingredient. For furtherinformation on Routes of Administration and Dosage Regimes the reader isreferred to Chapter 25.3 in Volume 5 of Comprehensive MedicinalChemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press1990.

The size of the dose for therapeutic or prophylactic purposes of acompound of the Formula I will naturally vary according to the natureand severity of the conditions, the age and sex of the animal or patientand the route of administration, according to well known principles ofmedicine. As mentioned above, compounds of the Formula I are useful intreating diseases or medical conditions which are due alone or in partto the effects of MCP-1 and/or RANTES, for example, rheumatoidarthritis.

In using a compound of the Formula (I) for therapeutic or prophylacticpurposes it will generally be administered so that a daily dose in therange, for example, 0.5 mg to 75 mg per kg body weight is received,given if required in divided doses. In general lower doses will beadministered when a parental route is employed. Thus, for example, forintravenous administration, a dose in the range, for example, 0.5 mg to30 mg per kg body weight will generally be used. Similarly, foradministration by inhalation, a dose in the range, for example, 0.5 mgto 25 mg per kg body weight will be used. Oral administration is howeverpreferred.

According to a further aspect of the present invention there is provideda compound of the formula (I) or a pharmaceutically acceptable salt orprodrug thereof, as defined hereinbefore for use in a method oftreatment of the human or animal body by therapy. Conveniently, theinvention provides a method of treating inflammatory disease byadministering a compound of formula (I) or a pharmaceutically acceptablesalt or prodrug thereof or a pharmaceutical composition thereof asdescribed above.

A further feature of the present invention is a compound of formula (I)and pharmaceutically acceptable salt or prodrug thereof, for use as amedicament.

Conveniently this is a compound of formula (I), or a pharmaceuticallyacceptable salt or prodrug thereof for use as a medicament forantagonising an MCP-1 (and/or RANTES) mediated effect in a warm-bloodedanimal such as a human being.

Thus according to a further aspect of the invention there is providedthe use of a compound of the formula (I), or a pharmaceuticallyacceptable salt or prodrug thereof, in the manufacture of a medicamentfor use in antagonising an MCP-1 (and/or RANTES) mediated effect in awarm-blooded animal such as a human being.

According to a further feature of the invention there is provided amethod of antagonising an MCP-1 (and/or RANTES) mediated effect in awarm-blooded animal, such as a human being, in need of such treatmentwhich comprises administering to said animal an effective amount of acompound of formula (I) or a pharmaceutically acceptable salt or prodrugthereon as defined hereinbefore.

Biological Testing.

The following biological test methods, data and Examples serve toillustrate the present invention.

Abbreviations:

ATCC American Type Culture Collection, Rockville, USA. BCABicinchroninic acid, (used, with copper sulphate, to assay protein) BSABovine Serum Albumin DMEM Dulbecco's modified Eagle's medium EGTAEthylenebis(oxyethylenenitrilo)tetraacetic acid FCS Foetal calf serumHEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]) HBSSHank's Balanced Salt Solution hMCP-1 Human Monocyte ChemoattractantProtein-1 PBS Phosphate buffered saline PCR Polymerase chain reaction

AMPLITAQ™ available from Perkin-Elmer Cetus, is used as the source ofthermostable DNA polymerase.

Binding Buffer is 50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% foetal calfserum, adjusted to pH 72 with 1 M NaOH.

Non-Essential Amino Acids (100× concentrate) is: L-Alanine, 890 mg/lL-Asparagine, 1320 mg/l; L-Aspartic acid, 1330 mg/l; L-Glutamic acid,1470 mg/l; Glycine, 750 mg/l; L-Proline, 1150 mg/l and; L-Serine, 1050mg/l.

Hypoxanthine and Thymidine Supplement (50× concentrate) is:hypoxanthine, 680 mg/l and; thymidine, 194 mg/l.

Penicillin-Streptomycin is: Penicillin G (sodium salt); 5000 units/ml;Streptomycin sulphate, 5000 μg/mL.

Human monocytic cell line THP-1 cells are available from ATCC, accessionnumber ATCC TIB-202.

Hank's Balanced Salt Solution (HBSS) was obtained from Gibco; see Proc.Soc. Exp. Biol. Med., 1949, 71, 196.

Synthetic cell culture medium, RPMI 1640 was obtained from Gibco; itcontains inorganic salts [Ca(NO₃)₂.4H₂O 100 mg/l; KCl 400 mg/l;MgSO₄.7H₂O 100 mg/l; NaCl 6000 mg/l; NaHCO₃ 2000 mg/l & Na₂HPO₄ (anhyd)800 mg/l)], D-Glucose 2000 mg/l, reduced glutathione 1 mg/l, amino acidsand vitamins.

FURA-2/AM is1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraaceticacid pentaacetoxymethyl ester and was obtained from Molecular Probes,Eugene, Oreg., USA.

Blood Sedimentation Buffer contains 8.5 g/l NaCl and 10 g/l hydroxyethylcellulose.

Lysis Buffer is 0.15 M NH₄CT, 10 mM KHCO₃, 1 mM EDTA

Whole Cell Binding Buffer is 50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 0.5%BSA, 0.01% NaN₃, adjusted to pH 7.2 with 1M NaOH.

Wash buffer is 50 mM HEPES 1 mM CaCl₂, 5 mM MgCl₂, 0.5% heat inactivatedFCS, 0.5 M NaCl adjusted to pH 7.2 with 1M NaOH.

General molecular biology procedures can be followed from any of themethods described in “Molecular Cloning—Laboratory Manual” SecondEdition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory,1989).

i) Cloning and Expression of hMCP-1 Receptor

The MCP-1 receptor B (CCR2B) cDNA was cloned by PCR from THP-1 cell RNAusing suitable oligonucleotide primers based on the published MCP-1receptor sequences (Charo et al., 1994, Proc. Natl. Acad. Sci. USA, 91,2752). The resulting PCR products were cloned into vector PCR-II™ (InVitrogen, San Diego, Calif.). Error free CCR2B cDNA was subcloned as aHind III-Not I fragment into the eukaryotic expression vector pCNA3 (InVitrogen) to generate pCDNA3/CC-CKR2A and pCDNA3/CCR2B respectively.

Linearised pCDNA3/CCR2B DNA was transfected into CHO-K1 cells by calciumphosphate precipitation (Wigler et al., 1979, Cell, 16, 777).Transfected cells were selected by the addition of Geneticin Sulphate(G418, Gibco BRL) at 1 mg/ml, 24 hours after the cells had beentransfected. Preparation of RNA and Northern blotting were carried outas described previously (Needham et al., 1995, Prot. Express. Purific.,6, 134). CHO-K1 clone 7 (CHO-CCR2B) was identified as the highest MCP-1receptor B expresser.

ii) Preparation of Membrane Fragments

CHO-CCR2B cells were grown in DMEM supplemented with 10% foetal calfserum, 2 mM glutamine, 1× Non-Essential Amino Acids, 1× Hypoxanthine andThymidine Supplement and Penicillin-Streptomycin (at 50 μgstreptomycin/ml, Gibco BRL). Membrane fragments were prepared using celllysis/differential centrifugation methods as described previously(Siciliano et al., 1990, J. Biol. Chem, 265, 19658). Proteinconcentration was estimated by BCA protein assay (Pierce, Rockford,Ill.) according to the manufacture's instructions.

iii) Assay

¹²⁵I MCP-1 was prepared using Bolton and Hunter conjugation (Bolton etal., 1973, Biochem. J., 133, 529; Amersham International plc].Equilibrium binding assays were carried out using the method of Ernst etal., 1994, J. Immunol., 152, 3541. Briefly, varying amounts of¹²⁵I-labeled MCP-1 were added to 7 μg of purified CHO-CCR2B cellmembranes in 100 μl of Binding Buffer. After 1 hour incubation at roomtemperature the binding reaction mixtures were filtered and washed 5times through a plate washer (Brandel MLR-96T Cell Harvester) using iceto cold Binding Buffer. Filter mats (Brandel GF/B) were pre-soaked for60 minutes in 0.3% polyethylenimine prior to use. Following filtrationindividual filters were separated into 3.5 ml tubes (Sarstedt No.55.484) and bound ¹²⁵I-labeled MCP-1 was determined (LKB 1277Gammamaster). Cold competition studies were performed as above using 100pM ¹²⁵I-labeled MCP-1 in the presence of varying concentrations ofunlabeled MCP-1. Non-specific binding was determined by the inclusion ofa 200-fold molar excess of unlabeled MCP-1 in the reaction.

Ligand binding studies with membrane fragments prepared from CHO-CCR2Bcells showed that the CCR2B receptor was present at a concentration of0.2 pmoles/mg of membrane protein and bound MCP-1 selectively and withhigh affinity (IC₅₀=110 pM, K_(d)=120 pM). Binding to these membraneswas completely reversible and reached equilibrium after 45 minutes atroom temperature, and there was a linear relationship between MCP-1binding and CHO-CCR2B cell membrane concentration when using MCP-1 atconcentrations between 100 pM and 500 pM.

Test compounds dissolved in DMSO (5 μl) were tested in competition with100 pM labelled MCP-1 over a concentration range (0.01-50 μM) induplicate using eight point dose-response curves and IC₅₀ concentrationswere calculated.

Compounds tested of the present invention had IC₅₀ values of 50 μM orless in the hMCP-1 receptor binding assay described herein.

b) MCP-1 mediated calcium flux in THP-1 cells

The human monocytic cell line THP-1 was grown in a synthetic cellstructure medium RPMI 1640 supplemented with 10% foetal calf serum, 6 mMglutamine and Penicillin-Streptomycin (at 50 μg streptomycin/ml, GibcoBRL). THP-1 cells were washed in HBSS (lacking Ca²⁺ and Mg²⁺)+1 mg/mlBSA and resuspended in the same buffer at a density of 3×10⁶ cells/ml.The cells were then loaded with 1 mM FURA-2/AM for 30 min at 37° C.,washed twice in HBSS, and resuspended at 1×10⁶ cells/ml. THP-1 cellsuspension (0.9 ml) was added to a 5 ml disposable cuvette containing amagnetic stirrer bar and 2.1 ml of prewarmed (37° C.) HBSS containing 1mg/ml BSA, 1 mM MgCl₂ and 2 mM CaCl₂. The cuvette was placed in afluorescence spectrophotometer (Perkin Elmer, Norwalk, Conn.) andpreincubated for 4 min at 37° C. with stirring. Fluorescence wasrecorded over 70 sec and cells were stimulated by addition of hMCP-1 tothe cuvette after 10 sec. [Ca²⁺]i was measured by excitation at 340 nmand 380 nm alternatively and subsequent measurement of the intensity ofthe fluorescence emission at 510 nm. The ratio of the intensities of theemitted fluorescent light following excitation at 340 nm and 380 nm,(R), was calculated and displayed to give and estimate of cytoplasmic[Ca²⁺]according to the equation:${\lbrack {Ca}^{2 +} \rbrack i} = {K_{d}\frac{( {R - {R\quad\min}} )}{( {{R\quad\max} - R} )}( {{Sf2}/{Sb2}} )}$where the K_(d) for FURA-2 Ca²⁺ complex at 37° C. was taken to be 224nm. R_(max) is the maximal fluorescence ratio determined after additionof 10 mM Ionomycin, R_(min) is the minimal ratio determined by thesubsequent addition of a Ca²⁺ free solution containing 5 mM EGTA, andSf2/Sb2 is the ratio of fluorescence value at 380 nm excitationdetermined at R_(min) and R_(max), respectively.

Stimulation of THP-1 cells with hMCP-1 induced a rapid, transient risein [Ca²⁺]i in a specific and dose dependant manner. Dose response curvesindicated an approximate EC₅₀ of 2 nm. Test compounds dissovled in DMSO(10 μl) were assayed for inhibition of calcium release by adding them tothe cell suspension 10 sec prior to ligand addition and measuring thereduction in the transmit rise in [Ca²⁺]i. Test compounds were alsochecked for lack of agonist activity by addition in place of hMCP-1.

c) hMCP-1 and RANTES mediated chemotaxis.

In vitro chemotaxis assays were performed using the human monocytic cellline THP-1. Cell migration through polycarbonate membranes was measuredby enumerating those passing through either directly by Coulter countingor indirectly by use of a colourimetric viability assay measuring thecleavage of a tetrazolium salt by the mitochondrial respiratory chain(Scudiero D. A. et al. 1988, Cancer Res., 48, 4827-4833).

Chemoattractants were introduced into a 96-well microtitre plate whichforms the lower well of a chemotaxis chamber fitted with a PVP-free 5 μmporesize polycarbonate adhesive framed filter membrane (NeuroProbe MBseries, Cabin John, Md. 20818, USA) according to the manufacturer'sinstructions. The chemoattractant was diluted as appropriate insynthetic cell culture medium, RPMI 1640 (Gibco) or supplemented with 2mM glutamine and 0.5% BSA, or alternatively with HBSS with Ca²⁺ and Mg²⁺without Phenol Red (Gibco) plus 0.1% BSA. Each dilution was degassedunder vacuum for 30 min and was placed (400 μl) in the lower wells ofthe chamber and THP-1 cells (5×10⁵ in 100 μl RPMI 1640+0.5% BSA) wereincubated in each well of the upper chamber. For the inhibition ofchemotaxis the chemoattractant was kept at a constant submaximalconcentration determined previously (1 nM MCP-1) and added to the lowerwell together with the test compounds dissolved in DMSO (final DMSOconcentration <0.05% v/v) at varying concentrations. The chamber wasincubated for 2 h at 37° C. under 5% CO₂. The medium was removed fromthe upper wells which were then washed out with 200 μl physiologicalsaline before opening the chamber, wiping dry the membrane surface andcentrifuging the 96-well plate at 600 g for 5 min to harvest the cells.Supernatant (150 μl was aspirated and 10 μl of cell proliferationreagent, WST-1,{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-phenyldisulfonate} plus an electron coupling reagent (Boehringer Mannheim,Cat. no. 1644 807) was added back to the wells. The plate was incubatedat 37° C. for 3 h and the absorbance of the soluble formazan product wasread on a microtitre plate reader at 450 nm. The data was input into aspreadsheet, corrected for any random migration in the absence ofchemoattractant and the average absorbance values, standard error of themean, and significance tests were calculated hMCP-1 inducedconcentration dependent cell migration with a characteristic biphasicresponse, maximal 0.5-1.0 nm.

In an alternative form of the above assay, fluorescently tagged cellscan be used in order to assist in end point detection. In this case, theTHP-1 cells used are fluorescently tagged by incubation in the presenceof 5 mM Calcein AM (Glycine, N,N′-[[3′,6′-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H), 9′-[9H]xanthene]-2′,7′-diyl]bis(methylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[(acetyloxy)methyl]ester;Molecular Probes) for 45 minutes in the dark. Cells are harvested bycentrifugation and resuspended in HBSS (without Phenol Red) with Ca²⁺,Mg²⁺ and 0.1% BSA. 50 μl (2×105 cells) of the cell suspension are placedon the filter above each well and, as above, the unit is incubated at37° C. for 2 hours under 5% CO₂. At the end of the incubation, cells arewashed off the upper face of the filter with phosphate buffered saline,the filter removed from the plate and the number of cells attracted toeither the underside of the filter or the lower well estimated byreading fluorescence at 485 nm excitation, 538 nm emission wavelengths(fmax, Molecular Devices). The data was input into a spreadsheet,corrected for any random migration in the absence of chemoattractant andthe average fluorescence values, standard error of the mean, percentageinhibition and IC₅₀ of compounds under test and significance tests canbe calculated. In addition to MCP-1 induced chemotaxis, this alternativeform of the assay was also used to measure inhibition of RANTES (2 nm)induced chemotaxis.

d) Binding to Human Peripheral Blood Mononuclear Cells (PBMCs)

i) Preparation of Human PBMCs

Fresh human blood (200 ml) was obtained from volunteer donors, collectedinto sodium citrate anticoagulant to give a final concentration of0.38%. The blood was mixed with Sedimentation Buffer and incubated at37° C. for 20 minutes. The supernatant was collected and centrifuged at1700 rpm for 5 minutes (Sorvall RT6000D). The pellet obtained wasresuspended in 20 ml RPMI/BSA (1 mg/ml) and 4×5 mls of cells werecarefully layered over 4×5 mls of Lymphoprepa (Nycomed) in 15 mlcentrifuge tubes. Tubes were spun at 1700 rpm for 30 minutes (SorvallRT6000D) and the resultant layer of cells was removed and transformed to50 ml Falcon tubes. The cells were washed twice in Lysis Buffer toremove any remaining red blood cells followed by 2 washes in RPMI/BSA.Cells were resuspended in 5 mls of Binding Buffer. Cell number wasmeasured on a Coulter counter and additional binding buffer was added togive a final concentration of 125×10⁷ PBMCs/ml.

ii) Assay

[¹²⁵I]MCP-1 was prepared using Bolton and Hunter conjugation (Bolton etal, 1973, Biochem J. 133, 529; Amersham International plc]. Equilibriumbinding assays were carried out using the method of Ernst et al., 1994,J. Immunol., 152, 3541. Briefly, 50 μl of ¹²⁵I-labeled MCP-1 (finalconcentration 100 pM) was added to 40 μl (5×10⁵ cells) of cellsuspension in a 96 well plate. Compounds, diluted in Whole Cell BindingBuffer from a stock solution of 10 mM in DMSO were added in a finalvolume of 5 μl to maintain a constant DMSO concentration in the assay of5%. Total binding was determined in the absence of compound.Non-specific binding was defined by the addition of 5 μl cold MCP-1 togive a final assay concentration of 100 nM. Assay wells were made up toa final volume of 100 μl with Whole Cell Binding Buffer and the platessealed. Following incubation at 37° C. for 60 minutes the bindingreaction mixtures were filtered and washed for 10 seconds using ice coldWash Buffer using a plate washer (Brandel MLR-96T Cell Harvester).Filter mats (Brandel GF/B) were pre-soaked for 60 minutes in 0.3%polyethylenimine plus 0.2% BSA prior to use. Following filtrationindividual filters were separated into 3.5 ml tubes (Sarstedt No.55.484) and bound ¹²⁵I-labeled MCP-1 was determined (LKB 1277Gammamaster).

Test compound potency was determined by assay in duplicate using sixpoint dose-response curves and IC₅₀ concentrations were determined.

No physiologically unacceptable toxicity was observed at the effectivedose for compounds tested of the present invention.

The invention is further illustrated, but not limited by the followingExamples in which the following general procedures were used unlessstated otherwise.

-   i) N,N-Dimethylformamide (DMF) was dried over 4 Å molecular sieves.    Anhydrous tetrahydrofuran (THF) was obtained from Aldrich SURESEAL™    bottles. Other commercially available reagents and solvents were    used without further purification unless otherwise stated. Organic    solvent extracts were dried over anhydrous MgSO₄.-   ii) ¹H, ¹³C and ¹⁹F NMR were recorded on Bruker WM200, WM250, WM300    or WM400 instruments using DMSO-d₆ with Me₄Si or CCl₃F as internal    standard as appropriate, unless otherwise stated. Chemical shifts    are quoted in d (ppm) and peak multiplicities are designated as    follows: s, singlet; d, doublet, dd, doublet of doublets; t,    triplet; dt, doublet of triplets; q, quartet; m, multiplet; br,    broad.-   iii) Mass spectra were recorded on VG 12—12 quadrupole, VG 70-250    SE, VG ZAB 2-SE or a VG modified AEI/Kratos MS9 spectrometers.-   iv) For TLC analysis, Merck precoated TLC plates (silica gel 60    F254, d=0.25 mm) were used.-   v) Flash chromatography was performed on silica (Merck Kieselgel:    Art 9385).

EXAMPLE 1 N-(3-methoxy-4-chlorobenzyl)-5-hydroxyindole-2-carboxylic Acid

Sodium hydroxide (2M, 1.9 ml) was added to a stirred solution of ethylN-(3-methoxy-4-chlorobenzyl)-5-acetoxyindole-2-carboxylate (305 mg) inmethanol (3 ml) and THF (3 ml). The reaction was stirred at roomtemperature for 18 hours. The reaction mixture was concentrated in vacuoand then diluted with water (5 ml). The solution was acidified by theaddition of aqueous hydrochloric acid (2M), extracted with ethylacetate, dried and evaporated. The residue was purified by columnchromatography eluting 20%-100% ethyl acetate to give the desiredproduct as a solid (177 mg, 70%) NMR (CD₃SOCD₃): d 3.95 (s, 3H), 5.95(s, 2H), 6.35 (d, 1H), 6.8 (dd, 1H), 6.9 (d, 1H), 7.0 (d, 1 μl), 7.1 (s,1H), 7.25 (d, 1H), 7.3 (d, 1H), 9.0 (s, 1H); m/z 332 (M+H⁺).

The procedure described in the above example was repeated using theappropriate indole ester as the starting material. Thus were obtainedthe compounds described below.

EXAMPLE 2 N-(3-chloro-4-methoxybenzyl)-5-hydroxyindole-2-carboxylic Acid

88% yield. NMR (CD₃SOCD₃): d 3.75 (s, 3H), 5.7 (s, 2H), 6.8 (dd, 1H),6.9-7.1 (m, 5H), 7.4 (d, 1H), 9.0 (bs, 1H); m/z 330 (M−H⁺).

EXAMPLE 3 N-(3-chloro-4-methylbenzyl)-5-hydroxyindole-2-carboxylic Acid

63% yield; m/z 314 (M−H⁺).

EXAMPLE 4 N-(3-nitro-4-methylbenzyl)-5-hydroxyindole-2-carboxylic Acid

5% yield; m/z 326 (M-H⁺).

EXAMPLE 5N-(3-chloro-4-trifluoromethoxybenzyl)-5-hydroxyindole-2-carboxylic Acid

82% yield. NMR (CD₃SOCD₃): d 5.8 (S, 2H), 6.8 (m, 1H), 6.98 (m, 2H),7.15 (s, 1H), 7.35 (m, 2H), 7.45 (m, 1H), 9.0 (S, 1H, 12.82 (S, 1); m/z384 (M−H⁺).

EXAMPLE 6 N-(3-nitro-4-chlorobenzyl)-5-hydroxyindole-2-carboxylic Acid

56% yield. NMR (CD₃SOCD₃): d 5.85 (S 2H), 6.85 (dd, 1H), 6.95 (d, 1),7.15 (M, 1H), 7.35 (d, 1H), 7.65 (d, 1H, 7.8 (d, 1H), 9.0 (S, 1H).

EXAMPLE 7 N-(3-fluoro-4-methylbenzyl)-5-hydroxyindole-2-carboxylic Acid

18% yield NMR (CD₃SOCD₃): d 5.7 (s, 2H), 6.7 (m, 3H), 6.9 (s, 10H, 7.1(m, 3H), 7.3 (m, 1H), 9.0 (s, 1H), 12.8 (s, 1H); m/z 298 (M−H⁺).

EXAMPLE 8 N-[(4-chloro-3-ethylphenyl)methyl]5-hydroxyindole-2-carboxylicAcid

Sodium hydroxide (2M, 1.8 ml) was added to a solution of ethylN-[(4-chloro-3-trimethylsilylethynylphenyl)methyl]-5-acetoxyindole-2-carboxylate(0.14 g) in methanol (5 ml) and the mixture was stirred for 3 hours. Themethanol was removed and the residue obtained was diluted with water (20ml) and extracted twice with ethyl acetate (20 ml each time). Theaqueous layer was acidified with aqueous hydrochloric acid (2M) andextracted with ethyl acetate (3×25 ml). The combined ethyl acetateextracts were washed with water (30 ml) and brine (30 ml) and dried(MgSO₄). The residue obtained on removal of the solvent was dissolved ina mixture of ethyl acetate and iso-hexane (1:1) and passed down a 5 gsilica Isolute column eluting with an ethyl acetate iso-hexane mixture(1:1) to give the title compound (65 mg). NMR (CD₃SOCD₃): d 4.5 (s, 1H),5.7 (s, 2H), 6.8 (m, 1H), 6.9 (m, 2H), 7.1 (m, 1H), 7.2 (s, 1H), 7.35(m, 1H), 7.4 (m, 2H), 9.0 (s, 1H); m/z 324.4 (M−H).

Preparation of Starting Materials

The starting materials for the Examples above are either commerciallyavailable or are readily prepared by standard methods from knownmaterials. For example the following reactions (Methods A-D) areillustrations but not limitations of the preparation of the startingmaterials used in the above reactions.

Method A

Ethyl 5-acetoxy-N-(3-methoxy-4-chlorobenzyl)indole-2-carboxylate i)Ethyl 5-hydroxyindole-2-carboxylate

Boron tribromide (64.58 g) was added dropwise to a stirred solution ofethyl 5-methoxyindole-2-carboxylate (20 g) in dichloromethane (1000 ml)at −78° C. under an atmosphere of argon. The reaction was allowed towarm to room temperature and stirred for a further 2 hours. The reactionwas poured into ice/s aqueous sodium hydrogen carbonate solution withstirring and extracted with ethyl acetate. Combined organic extractswere washed with saturated aqueous sodium hydrogen carbonate solution,water, aqueous saturated sodium chloride solution and dried. Thesolution was concentrated in vacuo and the residue was purified bycolumn chromatography using 0-60% diethyl ether, iso-hexane as eluent toyield product as a white solid (9.02 g, 48%). NMR: 1.31 (t, 3H), 4.29(q, 2H), 6.79 (dd, 1H), 6.90 (dd, 1H), 7.22 (d, 1H), 8.84 (s, 1H), 11.52(brs, 1H; m/z 206 M+H⁺).

ii) Ethyl 5-acetoxyindole-2-carboxylate

A stirred solution of ethyl 5-hydroxyindole-2-carboxylate (7.79 g) and4-dimethylaminopyridine (20 mg) in acetic anhydride (80 ml) was heatedat 80° C. for 4 hours. The reaction was concentrated in vacuo and theresidue was dissolved in ethyl acetate. Combined organic extracts werewashed with hydrochloric acid (2 M), saturated aqueous sodium hydrogencarbonate solution, water, aqueous saturated sodium chloride solutionand dried. The solution was concentrated in vacuo to yield the productas a yellow solid (9.39 g, 100%). NMR: 1.20 (t, 3H), 2.10 (s, 3H), 4.19(q, 2), 6.86 (dd, 1H), 6.97 (d, 1H), 7.20 (s, 1H), 7.29 (d, 1H); m/z 248(M+H⁺).

iii) Ethyl 5-acetoxy-N-(3-methoxy-4-chlorobenzyl)indole-2-carboxylate

To a solution of ethyl 5-acetoxyindole-2-carboxylate (283 mg) in DMF (6ml) was added sodium hydride (54 mg, 60% dispersion in oil). The mixturewas stirred for 30 minutes, a catalytic amount of potassium iodide and asolution of 3-methoxy-4-chlorobenzyl bromide (345 mg) in DMF (2 ml) wasadded. The mixture was stirred for 2 hours, quenched with water andextracted with ethyl acetate. The organic extracts were dried,evaporated and the resulting gum was purified by column chromatographyelutig with 20% ethyl acetate/isohexane to give the desired product asan oil which solidified on standing (310 mg, 63%). NMR (CDCl₃): d 1.4(t, 3H), 2.3 (s, 3H), 3.8 (s, 3H), 4.35 (q, 2H, 5.8 (s, 2H), 6.5 (dd,1H), 6.7 (d, 1H), 7.05 (dd, 1H), 7.2-7.4 (m, 4H); m/z 402 (M+H⁺).

The procedures described in Method A i)-iii) were repeated using theappropriate benzyl halide. Thus were obtained the compounds describedbelow.

Ethyl N-(3-chloro-4-methoxybenzyl)-5-hydroxyindole-2-carboxylate

58% yield; m/z 402 (MH⁺).

Ethyl N-(3-chloro-4-methylbenzyl)-5-hydroxyindole-2-carboxylate

73% yield; m/z 386 (MH⁺).

Ethyl N-(3-nitro-4-methylbenzyl)-5-hydroxyindole-2-carboxylate

41% yield; m/z 396 (MH⁺).

EthylN-(3-chloro-4-trifluoromethoxybenzyl)-5-hydroxyindole-2-carboxylate

86% yield NMR (CD₃SOCD₃): d 1.25 (t, 31), 2.25 (s, 3H), 4.25 (q, 2H),5.85 (s, 2M), 6.95 (m, 1H), 7.1 (m, 1H), 7.39 (m, 2H), 7.45 (m, 2H), 7.6(m, 1H; m/z 456 (MH⁺).

Ethyl N-(3-nitro-4-chlorobenzyl)-5-hydroxyindole-2-carboxylate

55% yield NMR (CD₃SOCD₃): d 1.39 (t, 3H), 2.31 (s, 3H), 4.32 (q, 2H),5.81 (s, 2H), 7.04-7.15 (m, 2H), 7.21-7.3 (m, 1H), 7.36-7.44 (m, 3H),7.62 (s, 1H).

Method B

3-Methoxy-4-chlorobenzyl Bromide (i)3-methoxy-4-chlorotoluene

To a solution of 2-chloro-5-methyl phenol (15.95 g) in acetone (200 ml)was added potassium carbonate (38 g) and dimethyl sulphate (11.7 ml).The mixture was refluxed for 3 hours and then filtered to give thedesired product which was used without further purification (16.95 g,98%). NMR (CDCl₃): d 2.35 (s, 3H), 3.9 (s, 3H), 6.7-6.8 (m, 2H),7.15-7.3 (m, 1H).

(ii)3-methoxy-4-chlorobenzyl Bromide

A mixture of 3-methoxy-4-chlorotoluene (9.62 g) and N-bromosuccinimide(12.05 g) was refluxed whilst being irradiated with light using aphotoflood lamp for 2 hours. The mixture was cooled, filtered andevaporated to give an oil which was purified by column chromatographyeluting with diethyl ether to give the desired product as an oil (14.67g, 95%). NMR (CDCl₃): d 3.9 (s, 3H), 4.45 (s, 2H), 6.9-7.0 (m, 2H),7.3-7.4 (m, 1H).

In a similar manner but starting from 2-chloro-4-methyl phenol wasprepared.

3-Chloro-4-methoxybenzyl Bromide

96% yield NMR (CDCl₃): d 3.9 (s, 3H), 4.45 (s, 2H), 6.9 (d, 1H), 7.25(dd, 1H), 7.4 (d, 1H).

Method C

3-Nitro-4-chlorobenzyl Bromide

To a solution of 3-nitro-4-chlorobenzyl alcohol (4.67 g) and triphenylphosphine (6.53 g) in dichloromethane (150 ml), cooled to 5° C. under anargon atmosphere, was added carbon tertrabromide (8.27 g). The resultingmixture was stirred at room temperature for 18 hours. The mixture wasconcentrated and purified by column chromatography eluting withiso-hexane rising to 20°ethyl acetate/isohexane to give the product as ayellow oil (5.39 g, 86%). NMR (CDCl₃): d 4.5 (s, 2H), 7.5 (s, 2H), 7.9(s, 1H).

Method D

EthylN-[(4-chloro-3-trimethylsilylethynylphenyl)methyl]-5-acetoxyindole-2-carboxylate(i)4-chloro-3-iodobenzyl Alcohol

Borane-THF complex (10 ml) was added dropwise over 20 minutes to asolution of 4-chloro-3-iodobenzoic acid (1.4 g) in THF (25 ml). Thereaction mixture was stirred for 2 hours and then cooled (ice bath) andmethanol (20 ml) was added cautiously. The solvent was removed and theresidue was dissolved in methanol (10 ml) and stirred with aq. 2M sodiumhydroxide (10 ml) for 2 hours. Ethyl acetate (50 ml) was added and themixture was washed with saturated aq. sodium bicarbonate solution (50ml). The aqueous extracts were washed with ethyl acetate (2×50 ml) andthe combined ethyl acetate extracts were washed with water (50 ml) andbrine (50 ml) and died. Removal of the solvent gave4-chloro-3-iodobenzyl alcohol (1.15 g). NMR (CD₃SOCD₃): d 4.45 (d, 2H),5.3 (t, 1H), 7.3 (m, 1H), 7.5 (m, 1H), 7.8 (s, 1H).

(ii)4-chloro-3-iodobenzyl Bromide

Triphenylphosphine (1.1 g) was added in portions to a solution of4-chloro-3-iodobenzyl alcohol (1.1 g) in dichloromethane (40 ml) at 0°C. and stirring was continued for 10 minutes. Carbon tetrabromide (1.5g) was then added in portions over 2 minutes, the reaction mixture wasallowed to warm to ambient temperature and was stirred for 15 hours. Thesolution was added directly to a silica charged chromatography columnand was eluted with dichloromethane to give 4-chloro-3-iodobenzylbromide (1.9 g). NMR (CD₃SOCD₃): d 4.6 (s, 2H), 7.5 (m, 2H), 7.6 (s,1H), 8.0 (m, 1H).

(iii) EthylN-[(4-chloro-3-iodophenyl)methyl]-5-acetoxyindole-2-carboxylate

Sodium hydride (0.23 g of a 60% dispersion in oil) was added to asolution of ethyl 5-acetoxyindole-2 carboxylate (1.29 g) andtetra-n-butylammonium iodide (10 mg) in anhydrous DMF (25 ml) under astream of argon. The reaction mixture was stirred for 15 minutes and asolution of 4-chloro-3-iodobenzyl bromide (1.9 g) in DMF (5 ml) wasadded. The mixture was stirred for 15 hours, then saturated aq. ammoniumchloride (5 ml) was added. The residue obtained on removal of thesolvent was diluted with saturated aq. ammonium chloride (50 ml) andextracted with ethyl actate (3×30 ml). The combined ethyl acetateextracts were washed with brine (100 ml) and dried. The residue obtainedon removal of the solvent was purified by chromatography on silicaeluting with a mixture of ethyl acetate and iso-hexane (1:9) to give thetitle compound (0.21 g). NMR (CD₃SOCD₃): d 1.1 (t, 3H), 2.2 (s, 3H), 4.3(m, 2H), 5.8 (s, 2H), 6.9 (m, 1H), 7.1 (m, 1H), 7.35 (m, 1H), 7.4 (m,2H), 7.6 (m 2H), 7.7 (m, 2H); m/z 497.5 (M+H).

(iv) EthylN-[(4-chloro-3-trimethylsilylethynylphenyl)methyl]-5-acetoxyindole-2-carboxylate

Trimethylsilylacetylene (114 il) was added to a degassed solution ofethyl N-[(4-chloro-3-iodophenyl) methyl]-5-acetoxyindole-2-carboxylate(0.2 g), bis(triphenylphosphine)palladium (II) chloride (2 mg),triethylamine (56 il) and copper (I) iodide (1 mg) in acetonitrile andthe mixture was stirred under an argon atmosphere for 12 hours. Afurther aliquot of bis(triphenylphosphine)palladium (II) chloride (2 mg)and trimethylsilylacetylene (114 il) was added and stirring continuedfor 2 hours. A further aliquot of trimethylsilylacetylene (114 il) wasadded and stirring was continued for 12 hours. A small amount of silicawas added to the reaction mixture and the solvent was evaporated. Theresidue was added to a 5 g silica Isolute column and eluted with anethyl acetate iso-hexane mixture (1:4) to give the title compound as acolourless oil (0.15 g). NMR (CD₃SOCD₃): d 0.0 (s, 9H), 1.1 (t, 31H),2.1 (s, 3H), 4.1 (m, 2H), 5.6 (s, 2H), 6.7 (m, 2H), 6.9 (m, 2H), 7.1 (m,1H), 7.2 (m, 2H), 7.3 (n, 1H), 7.4 (m, 1H); m/z 468.5 (M+H).

EXAMPLE 9

Pharmaceutical Compositions

This Example illustrates, but is not intended to limit, representativepharmaceutical dosage forms of the invention as defined herein (theactive ingredient being termed “Compound X”), for therapeutic orprophylactic use in humans:

EXAMPLE A

(a) Tablet I mg/tablet Compound X 100 Lactose Ph.Eur 182.75Croscarmellose sodium 12.0 Maize starch paste (5% w/v paste) 2.25Magnesium stearate 3.0 (b) Tablet II mg/tablet Compound X 50 LactosePh.Eur 223.75 Croscarmellose sodium 6.0 Maize starch 15.0Polyvinylpyrrolidone (5% w/v paste) 2.25 Magnesium stearate 3.0 (c)Tablet III mg/tablet Compound X 1.0 Lactose Ph.Eur 93.25 Croscramellosesodium 4.0 Maize starch paste (5% w/v paste) 0.75 Magnesium stearate 1.0(d) Capsule mg/capsule Compound X 10 Lactose Ph.Eur 488.5 Magnesium 1.5(e) Injection I (50 mg/ml) Compound X 5.0% w/v 1 M Sodium hydroxidesolution 15.0% v/v   0.1 M Hydrochloric acid to adjust pH to 7.6Polyethylene glycol 400 4.5% w/v Water for injection to 100% (f)Injection II (10 mg/ml) Compound X 1.0% w/v Sodium phosphate BP 3.6% w/v0.1 M Sodium hydroxide solution 15.0% v/v   Water for injection to 100%(g) Injection III (1 mg/ml buffered, to pH6) Compound X  0.1% w/v Sodiumphosphate BP 2.26% w/v Citric acid 0.38% w/v Polyethylene glycol 400 3.5% w/v Water for injection to 100% (h) Aerosol I mg/ml Compound X10.0 Sorbitan trioleate 13.5 Trichlorofluoromethane 910.0Dichlorodifluoromethane 490.0 (i) Aerosol II mg/ml Compound X 0.2Sorbitan trioleate 0.27 Trichlorofluoromethane 70.0Dichlorodifluoromethane 280.0 Dichlorotetrafluoroethane 1094.0 (j)Aerosol III mg/ml Compound X 2.5 Sorbitan trioleate 3.38Trichlorofluoromethane 67.5 Dichlorodifluoromethane 1086.0Dichlorotetrafluoroethane 191.6 (k) Aerosol IV mg/ml Compound X 2.5 Soyalecithin 2.7 Trichlorofluoromethane 67.5 Dichlorodifluoromethane 1086.0Dichlorotetrafluoroethane 191.6 (l) Ointment ml Compound X 40 mg Ethanol300 μl Water 300 μl 1-Dodecylazacycloheptan-2-one 50 μl Propylene glycolto 1 ml Note: Compound X in the above formulations may comprise acompound as illustrated in Examples herein. The above formulations maybe obtained by conventional procedures well known in the pharmaceuticalart. The tablets (a)- (c) may be enteric coated by conventional means,for example to provide a coating of cellulose acetate phthalate. Theaerosol formulations (h)-(k) may be used in conjunction with standard,metered dose aerosol dispensers, and the suspending agents sorbitantrioleate and soya lecithin may be replaced by an alternative suspendingagent such as sorbitan monooleate, sorbitan # sesquioleate, polysorbate80, polyglycerol oleate or oleic acid.

1. A compound of the formula (I):

wherein: R¹ is hydrogen, halo, methyl, ethyl, or methoxy; R² ishydrogen, halo, methyl, ethyl, or methoxy; R³ is a halo group, loweralkyl, lower alkenyl, lower alkynyl, alkoxy, trifluoromethyl, nitro,cyano, trifluoromethoxy, C(O)R⁷, or S(O)_(n)R⁷ where n is 0, 1, or 2 andR⁷ is an alkyl group; R⁴ is a halo, trifluoromethyl, methylthio,methoxy, trifluoromethoxy, lower alkyl, lower alkenyl, or lower alkynyl;R⁵ is hydrogen, halo, cyano, lower alkyl, lower alkenyl, lower alkynyl,or COR⁸ where R⁸ is lower alkyl; and R⁶ is hydrogen, halo, lower alkyl,lower alkenyl, lower alkynyl, or COR⁹ where R⁹ is lower alkyl; providedthat when R¹ is hydrogen, halo, or methoxy; R² is hydrogen, halo,methyl, ethyl, or methoxy; and R⁵ and R⁶ are both hydrogen; then a) whenone of R³ or R⁴ is chloro, fluoro, or trifluoromethyl, then the other isnot halo or trifluoromethyl; b) when one of R³ or R⁴ is chloro, theother is not methyl; and c) when R³ is trifluoromethyl, R⁴ is notmethyl; or a pharmaceutically acceptable salt or prodrug thereof.
 2. Acompound according to claim 1, wherein in the formula (I): R¹ ishydrogen or a halo group; R² is hydrogen or a halo group; R³ is a halogroup, nitro or alkoxy; R⁴ is a halo group, lower alkyl, methoxy, ortrifluoromethoxy; and R⁵ and R⁶ are hydrogen.
 3. A compound according toclaim 1, wherein in the formula (I): R¹ is hydrogen or a halo group; R²is hydrogen or a halo group; R³ is trifluoromethyl; R⁴ is methylthio,methoxy, trifluoromethoxy, lower alkyl or lower alkynyl; and R⁵ and R⁶are hydrogen.
 4. A compound according to claim 1, wherein in the formula(I): R¹ is hydrogen or a halo group; R² is hydrogen or a halo group; R³is lower alky, lower alkenyl, lower alkynyl, alkoxy, nitro, cyano,trifluoromethoxy, C(O)R⁷, or S(O)nR⁷ where n is 0, 1 or 2 and R⁷ is analkyl group; R⁴ is a halo group or trifluoromethyl; and R⁵ and R⁶ arehydrogen.
 5. A compound according to claim 1 which is a compound of theformula (IA):

wherein: R¹ is hydrogen, halo, methyl, ethyl, or methoxy; R² ishydrogen, halo, methyl, ethyl, or methoxy; R³′ is chloro, lower alkyl,lower alkenyl, lower alkynyl, alkoxy, trifluoromethyl, nitro, cyano,trifluoromethoxy, C(O)R⁷, or S(O)nR⁷ where n is 0, 1, or 2 and R⁷ is analkyl group; and R⁴′ is a halo group, methylthio, methoxy,trifluoromethoxy, or methyl; provided that when R^(3′) istrifluoromethyl, R^(4′) is not trifluoromethyl or halo; or apharmaceutically acceptable salt or prodrug thereof.
 6. A compoundaccording to claim 5, wherein in the formula (IA): R¹ and R² arehydrogen; R³ is methoxy, chloro or nitro; and R⁴ is chloro, methyl,methoxy or trifluoromethoxy.
 7. A compound according to claim 1 which isany of the following:N-(3-methoxy-4-chlorobenzyl)-5-hydroxyindole-2-carboxylic acid;N-(3-chloro-4-methoxybenzyl)-5-hydroxyindole-2-carboxylic acid;N-(3-nitro-4-methylbenzyl)-5-hydroxyindole-2-carboxylic acid;N-(3-chloro-4-trifluoromethoxybenzyl)-5-hydroxyindole-2-carboxylic acid;N-(3-nitro-4-chlorobenzyl)-5-hydroxyindole-2-carboxylic acid;N-(3-fluoro-4-methylbenzyl)-5-hydroxyindole-2-carboxylic acid; orN-[(4-chloro-3-ethynylphenyl)methyl]-5-hydroxyindole-2-carboxylic acid.8. A process for preparing a compound according to claim 1 or apharmaceutically acceptable salt or prodrug thereof, which processcomprises: (a) reacting a compound of formula (II):

where R¹, R², R⁵, and R⁶ are as defined in claim 1; R^(a) is carboxy ora protected form thereof, and R^(b) is hydrogen or a suitable hydroxylprotecting group, with a compound of formula (III):

where R³ and R⁴ are defined as in claim 1; and L is a displaceablegroup; and optionally thereafter: (b) (i) converting a resultingcompound of the formula (1) into another compound of the formula (I);(ii) removing any protecting groups; or (iii) forming a pharmaceuticallyacceptable salt or prodrug thereof.
 9. A method of treating arthritis,glomerular nephritides, lung fibrosis, restinosis, alveolitis, asthma,atherosclerosis, psoriasis, hypersensitivity of the skin, inflammatorybowel disease, multiple sclerosis, brain trauma, stroke, reperfusioninjury, ischemia, myocardial infarction or transplant rejection in ananimal, comprising administering an effective amount of the compoundaccording to any one of claims 1 to 7 or a pharmaceutically acceptablesalt or prodrug thereof.
 10. A pharmaceutical composition comprising acompound according to any one of claims 1 to 7 or a pharmaceuticallyacceptable salt or prodrug thereof, in association with apharmaceutically acceptable excipient or carrier.
 11. A method ofmanufacturing a medicament, comprising providing a compound according toany one of claims 1 to 7 or a pharmaceutically acceptable salt orprodrug thereof.
 12. A method of treating inflammatory disease whichcomprises administering to a host in need of such treatment an effectiveamount of the compound according to any one of claims 1 to 7 or apharmaceutically acceptable salt or prodrug thereof.
 13. A method oftreating inflammatory disease which comprises administering to a host inneed of such treatment an effective amount of the pharmaceuticalcomposition according to claim
 10. 14. A method of treating arthritis,glomerular nephritides, lung fibrosis, restinosis, alveolitis, asthma,atherosclerosis, psoriasis, hypersensitivity of the skin, inflammatorybowel disease, multiple sclerosis, brain trauma, stroke, reperfusioninjury, ischemia, myocardial infarction or transplant rejection in ananimal, comprising administering an effective amount of a pharmaceuticalcomposition according to claim
 10. 15. A compound according to claim 1,wherein R¹ is hydrogen or halo.
 16. A compound according to claim 1,wherein R² is hydrogen or halo.
 17. A compound according to claim 1,wherein R³ is selected from halo, nitro or alkoxy.
 18. A compoundaccording to claim 1, wherein R³ is trifluoromethyl and R⁴ ismethylthio, methoxy, trifluoromethoxy, or alkynyl.
 19. A compoundaccording to claim 1, wherein R⁴ is halo, methoxy, or trifluoromethoxy.20. A compound according to claim 1, wherein R⁴ is halo ortrifluoromethyl and R³ is alkenyl, alkynyl, alkoxy, nitro, cyano,trifluoromethoxy, C(O)R⁷, or S(O)_(n)R⁷ where R⁷ and n are as defined inclaim
 1. 21. A compound according to claim 1, wherein R⁵ is hydrogen.22. A compound according to claim 1, wherein R⁶ is hydrogen.